Abstract:

The invention discloses a porous composite biomaterial comprising of
poly(γ-glutamic acid)-g-chondroitin sulfate (γ-PGA-g-CS)
copolymer and poly(ε-caprolactone). The composite biomaterial
provides a three-dimensional microenvironment for using as a scaffold for
tissue engineering and for supporting the attachment and proliferation of
cells. The invention also discloses a method of producing a porous
composite biomaterial.

Claims:

2. The copolymer of claim 1, wherein weight percentage of said
poly(γ-glutamic acid) in said copolymer is in range of 1% to 50%,
and weight percentage of said chondroitin sulfate in said copolymer is in
range of 1% to 50%.

3. The copolymer of claim 1, wherein molar ratio of said
poly(γ-glutamic acid) to said chondroitin sulfate is about 1:0.5.

4. The copolymer of claim 1, wherein said cross-linking agent includes
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or
N,N'-dicyclohexylcarbodiimide (DCC), and weight percentage of said
cross-linking agent is in range of 1% to 200%.

5. The copolymer of claim 4, wherein molar ratio of said cross-linking
agent to said poly(γ-glutamic acid) is about 1:1.5.

6. A porous composite biomaterial, comprising:a copolymer;
andpoly(ε-caprolactone) (PCL);wherein weight percentage of said
copolymer in said porous composite biomaterial is in range of 1% to 70%,
and said copolymer is synthesized by cross-linking reaction between
poly(γ-glutamic acid) and chondroitin sulfate via a cross-linking
agent.

7. The porous composite biomaterial of claim 6, wherein weight percentage
of said poly(γ-glutamic acid) in said copolymer is in range of 1%
to 50%, and weight percentage of said chondroitin sulfate in said
copolymer is in range of 1% to 50%.

8. The porous composite biomaterial of claim 6, wherein said cross-linking
agent includes 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or
N,N'-dicyclohexylcarbodiimide (DCC), and weight percentage of said
cross-linking agent is in range of 1% to 200%.

9. The porous composite biomaterial of claim 6, wherein said porous
composite biomaterial may be utilized for scaffold of chondrocyte
culture.

10. The porous composite biomaterial of claim 6, wherein hydrophilicity of
said porous composite biomaterial increases as the content of said
copolymer increases.

11. The porous composite biomaterial of claim 6, wherein adsorption
ability of cells and tissues to said porous composite biomaterial
increases as content of said copolymer increases.

12. The porous composite biomaterial of claim 6, wherein degradability of
said porous composite biomaterial increases as content of said copolymer
increases.

13. A method of producing porous composite biomaterial, which
comprising:cross-linking segments of poly(γ-glutamic acid) and
chondroitin to synthesize a copolymer via a cross-linking agent;forming a
solution by dissolving and mixing said copolymer and
poly(ε-caprolactone) in solvent; andforming said porous composite
biomaterial by drying and shaping said solution;wherein weight percentage
of said copolymer in said porous composite biomaterial is in range of 1%
to 70%.

14. The method of producing porous composite biomaterial of claim 13,
wherein weight percentage of said poly(γ-glutamic acid) in said
copolymer is in range of 1% to 50%, and weight percentage of said
chondroitin sulfate in said cpolymer is in range of 1% to 50%.

15. The method of producing porous composite biomaterial of claim 13,
wherein said cross-linking agent includes
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) or
N,N'-dicyclohexylcarbodiimide (DCC), and weight percentage of said
cross-linking agent is in range of 1% to 200%.

16. The method of producing porous composite biomaterial of claim 13,
further comprising producing scaffold for chondrocyte culture by said
porous composite biomaterial.

17. The method of producing porous composite biomaterial of claim 13,
wherein hydrophilicity of said porous composite biomaterial increases as
the content of said copolymer increases.

18. The method of producing porous composite biomaterial of claim 13,
wherein degradability of said porous composite biomaterial increases as
content of said copolymer increases.

19. The method of producing porous composite biomaterial of claim 13,
further comprising:adding salts into said solution before drying and
shaping said solution; andremoving said salts from said solution after
drying and shaping said solution for forming three-dimensional porous
structures of said porous composite biomaterial; wherein particle size of
said salts is in range of 100 to 450 μm.

Description:

[0001]The present invention is generally related to the field of a porous
composite biomaterial, more particularly, to a porous composite
biomaterial of scaffold for tissue engineering.

DESCRIPTION OF THE PRIOR ART

[0002]Though the medical science has progressed over the years, issues
regarding tissue lesion or disability are mainly still managed by
conventional surgical operations of organ transplants. Because of the
problems of the source shortage and the immunological rejection, the
development of tissue engineering is widely carried out attempting to
solve the problems by culturing the human organs and tissues artificially
for patients all over the world. The basic concept of tissue engineering
is to develop the substitutes of the wounded tissues based on biological
and engineering technologies and maintain the normal operations of one
human body by acquiring a few cells from the human body, implanting the
cells into the plane or the steric materials to culture more cells in
vitro, and implanting the cultured cells into the human body to make up
or replace the wounded tissues of the human body. Because the cells are
sourced from the same human body, the immune responses may be avoided.
The base material of the cell culture scaffold is important relating to
the cell culture processes. The materials of the scaffold should not only
provide the spaces for cells to attach and to culture, but also leave the
structural messages to the cells to proliferate and differentiate to the
needed type of cells. Therefore, the materials for culturing the cells of
tissues should generally fulfill the following requirements: [0003]1.
No cytotoxicity and compatible with cells: It allows the cells to attach
on the material and to proliferate. [0004]2. High porosity: Without
enough space, the cells can proliferate only on the surface of the
material but not deep inside the material. For culturing the tissues
having three-dimensional structures, it is best to use the
three-dimensional materials with interconnected porous structures to
facilitate the conveyance of the nutrition and the wastes related to the
cells. [0005]3. Appropriate mechanical strength: The material should at
least be able to afford the weight of the new generated cartilage tissues
and have the properties of the polymers which is easy to be processed to
fit the different requirements of the patients. [0006]4.
Biodegradability: The material provides a temporary scaffold for cells to
proliferate, but they should be biodegradable and be replaced by the new
generated cells and the extracelluar matrices (ECM) of such cells to form
a whole new tissue.

[0007]The base materials of making the scaffolds generally comprise the
natural materials and the artificial synthetic materials, and owning
their advantages and disadvantages, respectively. While the natural
materials which acquired from the nature sources are quite compatible
with the cells, their applications are limited due to their low degrade
rate and mechanical strength. In contrast, the artificial synthetic
materials have relative good mechanical strength and controllable degrade
rate, but they have relative bad cell compatibility. The United States
Patent Application 2004/0166169 "Porous Extracellular Matrix Scaffold and
Method" discloses a material used for porous scaffold and the relative
method, but it did not mention the copolymer disclosed in the present
invention. The copolymer will be described in detail in this
specification.

[0008]The above-mentioned natural materials include collagen, alginate,
poly(γ-glutamic acid) (γ-PGA), chitosan, polysaccharides,
etc. Among the variety of natural copolymers, poly(γ-glutamic acid)
is a totally biodegradable natural copolymer. Its molecular weight is in
the range of about 100 kDA to 1000 kDA. It has good biocompatibility,
biodegradability, water absorption ability, and water permeability, so
that it is a good biomedical material. The poly(γ-glutamic acid) is
formed from the polymerization of the glutamic acids. Because it is
comprised of unitary amino acid unit, it has the properties of
non-toxicity, biocompatibility, and biodegradability. Besides, when the
hydrogen atom on the "α-COOH" position of the poly(γ-glutamic
acid) is displaced, its water absorption ability is improved, and it is
contributive to increase the hydrophilicity of the scaffold made of
poly(γ-glutamic acid). Adding the water solube carbodiimides as
cross-linking agents, poly(γ-glutamic acid) and fiber glue are
utilized to synthesize a new biological gel which has low cytotoxicity
for human body. Furthermore, the complex salt of poly(γ-glutamic
acid) (with magnesium, calcium, barium, sodium, lithium, etc) is widely
applied in the field of biomedical material, such as the materials of the
surgical suture line, the materials of wound care dressings, the
materials of wound healings, and the materials of hemostasis. Chondroitin
sulfate (CS), one of the common mucopolysaccharides in human body, is one
of the components of the extracellular matrix of cartilaginous tissue. It
has been proved effective in proliferating the cartilaginous tissue and
inhibiting the immune inflammation, and it make the cartilaginous tissue
which is without vessels and nerves take the nutrition in and metabolize
the waste. The chondroitin sulfate, with its low degree of crystallinity,
enables it to sustain the compression force and help to against the
repeated stretching motions. Such natural polymers have good
biocompatibility with cartilaginous cells and greatly improve the
biological properties of the scaffold made of such materials.

[0009]There are various artificial synthetic polymeric materials, and they
can be roughly divided into two groups, the biodegradable group and the
non-biodegradable group. In biomedical applications, the biodegradable
group is more important. After implanting the polymers into the human
body, the polymers can be decomposed into non-toxic small molecules by
the microorganisms or the enzymes in the human body and be filtered by
the kidney or be excreted by the process of metabolism. Thus, the
patients can avoid suffering multiple surgical operations and therefore
reduce the pain. These polymers mainly comprise carbon chains, further
comprising different structures such as ester bond, ether bond, amino
group, etc. There are plenty of biodegradable materials, but when
applying to biomedical field, the chosen materials should be degraded
within an acceptable time range and be mainly small molecules after
degrading. Thus, the chosen materials can easily be absorbed or
decomposed by creatures, and they can be excreted into the environment
and be recycled in the environment to reduce the impacts of the
environment. Polyester polymers play a major part in biodegradable
polymeric materials, because their ester bonds can easily be broken by
hydrolysis to form lactic acids that can be absorbed by a creature,
thereby transforming the lactic acids into carbon dioxides and water
molecules via metabolism inside the body of creature, and then excreted
from the creature. Therefore, such polyester polymers are widely used in
the field of biomedical materials. Poly(ε-caprolactone) (PCL) is
a common aliphatic polyester which is formed from the ring opening
polymerization of ε-caprolactones. Poly(ε-caprolactone)
has properties such as good mechanical strength, biocompatibility,
biodegradability, and permeability. It is widely used in the fields of
biomedical engineering and been certified by Food and Drug Administration
(FDA) that it can be applied to human bodies. So,
poly(ε-caprolactone) has high potential to be the base material
of issue engineering. However, poly(ε-caprolactone) has some
disadvantages such as poor hydrophilicity, low cell adsorption ability,
and slow degradation rate, thereby having negative effects on cell
proliferation to be the base material of tissue culture. The academia and
industries have paid efforts to develop the high potential base materials
based on the poly(ε-caprolactone), trying to keep the advantages
of poly(ε-caprolactone) and mend the foregoing disadvantages. The
U.S. Pat. No. 5,932,539 "Biodegradable Polymer Matrix for Tissue Repair"
disclosed a biodegradable polymer material for tissue repairing. It
disclosed the use of chondroitin sulfate and several polyamino acids, but
it did not mention the copolymers for improving the properties of
poly(ε-caprolactone).

SUMMARY OF THE INVENTION

[0010]The present invention discloses a porous composite biomaterial for
tissue engineering and production method of the same.

[0011]In one aspect of the present invention, a new copolymer is
synthesized by way of chemical bonding. The copolymer includes
poly(γ-glutamic acid)-g-chondroitin sulfate (γ-PGA-g-CS)
copolymer which comprising poly(γ-glutamic acid) and chondroitin
sulfate. Mixing the salt particle type poly(γ-glutamic
acid)-g-chondroitin sulfate copolymer with poly(ε-caprolactone)
to make poly(γ-PGA)-g-CS/PCL composite biomaterials, a material of
scaffold with similar properties of the extracelluar matrices is
obtained. The scaffold made of poly(γ-PGA)-g-CS/PCL composite
biomaterials have good hydrophilicity, cell adsorption ability, and
degradability. So, it should be a better scaffold material for tissue
culture than only poly(ε-caprolactone).

[0012]The poly(γ-glutamic acid)-g-chondroitin sulfate copolymer
according to the present invention includes segments of
poly(γ-glutamic acid) and chondroitin sulfate, and it is produced
by the cross-linking reaction between the mixed poly(γ-glutamic
acid) and chondroitin sulfate with the existence of the cross-linking
agents and organic solvents.

[0013]The method of producing the porous composite biomaterial according
to the present invention comprises mixing and dissolving the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer and
poly(ε-caprolactone) in solvents to get a solution, drying the
solution and shaping, and getting the porous composite biomaterial. The
weight percentage of the poly(γ-glutamic acid)-g-chondroitin
sulfate copolymer in this biomaterial is in the range of about 1% to 70%,
and the properties of the biomaterial changes with the different weight
percentage of the poly(γ-glutamic acid)-g-chondroitin sulfate
copolymer.

[0014]The porous composite biomaterial according to the present invention
has improved hydrophilicity, cell adsorption ability, and degradability,
which is better than only poly(ε-caprolactone) material.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 illustrates the diagram of the reaction mechanism of
synthesizing the poly(γ-glutamic acid)-g-chondroitin sulfate
copolymer of the preferred embodiment of the present invention.

[0016]FIG. 2 illustrates the 1H-NMR diagram of the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer of the
preferred embodiment of the present invention.

[0018]FIG. 4 illustrates the ESCA diagram of the scaffold made of the
porous composite biomaterial of the preferred embodiment of the present
invention.

[0019]FIG. 5 illustrates the ESCA diagram of the scaffold made of the
porous composite biomaterial of the preferred embodiment of the present
invention.

[0020]FIG. 6 illustrates the ESCA diagram of the scaffold made of the
porous composite biomaterial of the preferred embodiment of the present
invention.

[0021]FIG. 7 illustrates the TEM diagram of the scaffold made of the
porous composite biomaterial of the preferred embodiment of the present
invention.

[0022]FIG. 8 illustrates the diagram of the weight loss of the various
scaffolds via hydrolysis of the preferred embodiment of the present
invention.

[0023]FIG. 9 illustrates the analysis diagram of the cytotoxicity of the
various scaffolds of the preferred embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENT

[0024]The invention discloses a new poly(γ-glutamic
acid)-g-chondroitin sulfate copolymer produced by chemical synthesis
processes which graft the poly(γ-glutamic acid) onto the
chondroitin sulfate to form a graft copolymer. The chondroitin sulfate, a
natural polysaccharide polymer, is relatively abundant around the
cartilage in the human body, and it plays the key role in induction of
the chondrocytes (cartilage cells). And then, because
poly(γ-glutamic acid) owns great hydrophilicity, the
poly(γ-glutamic acid) is mixed with hydrophobic
poly(ε-caprolactone) for the good of combining both their
advantages in applying to the cartilage tissue culture in the field of
tissue engineering. Combining the chondroitin sulfate, the major
component of the extracellular matrix of cartilage tissue cells, into the
composite biomaterial to form the three-dimensional porous scaffold, the
scaffold is obtained and it provides a similar microenvironment to the
original cell. Then, a faster cell proliferating rate and more
extracellular matrices on the scaffold is obtained when culturing
chondrocytes.

[0025]Followings are the exemplary preferred embodiments of the present
invention:

Preparation of poly(γ-glutamic acid)-g-chondroitin sulfate copolymer

[0026]In this embodiment of the present invention, poly(γ-glutamic
acid) (γ-PGA), 4-dimethylaminopyridine (DMAP) and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) are put into dimethyl
sulfoxide (DMSO) as a solution, and the solution is oscillated within the
ultrasonic oscillator. Wherein the EDC can be replaced by
N,N'-dicyclohexylcarbodiimide (DCC). On the other hand, adequate amount
of chondroitin sulfates are dissolved in the water as another solution.
Then the two solutions are mixed, and the range of the ratio of dimethyl
sulfoxide solution to water solution is as 5:5 to 9:1. Wherein the molar
ratio of poly(γ-glutamic acid), chondroitin sulfate, and
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide is 1:0.5:1.5. The solution
is put in the sample bottle and stirred periodically, wherein the range
of the period is between 1 to 48 hours. After the solution is added into
excess of acetone with instillation, the product of precipitation is
extracted by aspirator filtration. The precipitation is dissolved into
phosphate buffer solution (PBS), wherein the molecular weight cut-off is
in the range of 10,000 to 100,000. It is dialyzed in the deionized water
for two days, and the water is changed every twelve hours. Then the
product is obtained after dialysis. The following steps include putting
the product in the centrifuge bottle and removing the liquid by
lyophilization method, then a drying sequence is performed to dry the dry
product. Then, the surface modification is performed on the product,
wherein the surface modification comprises poly(γ-glutamic acid)
and chondroitin sulfate with 1,6-hexanediamine. First, overdose of
1,6-hexanediamine is added into the solution to react under room
temperature around 1 to 48 hours. The solution after reacting is put into
the dialyzing membrane, wherein the molecular weight cut-off is 3,500 to
100,000. The solution is dialyzed in the deionized water for two days,
and the water is changed every 12 hours. Then the product is obtained
after dialysis. The reaction mechanism of the above-mentioned graft
copolymerization is shown in FIG. 1. And then the nuclear magnetic
resonance (NMR) is used to verify its structure, as shown in FIG. 2. The
NMR diagram shows that the chondroitin sulfate has been successfully
grafted onto the poly(γ-glutamic acid) via its hydroxyl group and
formed poly(γ-glutamic acid)-g-chondroitin sulfate copolymer. FIG.
3 illustrates the structure and labels corresponding to 1H-NMR
diagram in FIG. 2. Furthermore, regarding to the above-mentioned
copolymer, the average molecular weight of chondroitin sulfate is in the
range of about 2,000 to 50,000, and the average molecular weight of
poly(γ-glutamic acid) is in the range about 2,000 to 500,000.

Preparation of Porous Scaffold

[0027]Due to the good hydrophilicity of poly(γ-glutamic acid) and
chondroitin sulfate, above material is mixed with hydrophobic
poly(ε-caprolactone) and get the scaffold which provides a
similar microenvironment to the original cell. Faster cell proliferating
rates are achieved and more extracellular matrices are generated on the
scaffold in the processes of chondrocyte culture. In this embodiment of
the present invention, an appropriate amount of copolymer is taken and
dissolved into cosolvents (the range of the ratio of water and dimethyl
sulfoxide is 5:5 to 9:1). Stirring the solution with high speed,
subsequently, the chloroform is added into the solution. The appropriate
amount of poly(ε-caprolactone) is dissolved into the solution to
prepare various polymer solution with weight percentage of about 15%.
When homogeneously dissolved, the sieved salts is put with the diameter
100 to 450 μm into the solution rapidly and stir it thoroughly. After
that, the solution is poured into a Teflon mold for shaping and drying,
and it is put into cosolvent (the range of the ratio of water and
dimethyl sulfoxide is 5:5 to 9:1) with stirring. The solvent is changed
every 12 hours till all the salts are removed, following by freezing and
drying it to get the material. Subsequently, the excess part of the
material is cut and then the scaffold for tissue culture is obtained.

[0028]The electron spectroscopy for chemical analysis (ESCA) is introduced
to measure and verify the structure of the above-mentioned porous
scaffold. The ESCA emits X ray to excite and ionize the electrons from
the inner-shell orbitals of the atoms of the surface of object to get
photoelectrons. By determining the kinetic energies of these
photoelectrons, the electron binding energies is obtained. The electron
binding energies are dependent on the different properties of the atoms,
making ESCA useful to identify the species and chemical states of the
surface of object. ESCA is applied to verify the scaffold made of the
porous materials of the present invention to check if there are actually
the wanted copolymers inside the material. The result of ESCA verified
the scaffold made of porous material contains
poly(ε-caprolactone) (--C1S, 283 eV; --O1S, 531 eV; O
KLL, 997 eV), poly(γ-glutamic acid) (--N1S, 403 eV), and
chondroitin sulfate (--S2P3,2, 172.2 eV). The results are shown in
FIG. 4, FIG. 5, and FIG. 6.

[0029]In addition, the field emission scanning electron microscope
(FE-SEM) is utilized to observe the surface structure of the scaffold
made of porous material, and the result is shown in FIG. 7.

Analysis of the Mechanical Properties of the Scaffold

[0030]In another embodiment of the present invention, the scaffold
originally immersed in the cell culture broth is taken out. Then the
excess moisture on the scaffold is wiped off, and the scaffold is put on
the center of the platform of compression mold. Then, with the
compression rate of one millimeter per minute, the Universal Tensile
Tester (Instron®) is used to commence the compression test. After
inputting the required parameters, the compression modulus of the
scaffold is gotten as shown in Table 1. From the experimental data, it is
obvious that the compression strength of the scaffold decreases as the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer content
increases. This is due to the difference of molecular weights between the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer and
poly(ε-caprolactone) in the scaffold of the present invention.
Therefore, the whole compression strength decreases as the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer content in
this scaffold increases.

[0031]In this embodiment of the present invention, the degradability of
the prepared scaffold made of porous material is analyzed via the
percentage of weight change. At first, the dry weight W0 of the
scaffold is measured, and then the scaffold is put into a phosphate
buffer solution at 37° C. for hydrolytic reaction and changing the
buffer solution every three days. The scaffold is taken out of the
solution periodically for weighting. Before weighting, the scaffold is
put into the double distilled water with ultrasonic oscillation to remove
the residue of salts from the buffer solution, and then the scaffold is
dehydrated with the absolute alcohol and measure the weight at that time
W1. Then, the percentage of weight change is

( W 0 - W 1 ) W 0 × 100 % . ##EQU00001##

[0032]The result is shown in FIG. 8, wherein the
poly(ε-caprolactone) content in the sample "PCL" is 100%, while
the ratio of poly(γ-glutamic acid)-g-chondroitin sulfate copolymer
and poly(ε-caprolactone) in "R10P90" is 10:90, and the ratio is
30:70 in "R30P70". From the experimental result, it may be concluded that
the degradability of scaffold increases as the poly(γ-glutamic
acid)-g-chondroitin sulfate copolymer content increases and proved that
the degradability of the scaffold may be improved by adding the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer.

The Water Absorption and the Porosity Test

[0033]In this embodiment of the present invention, the tests for water
absorption and the porosity of the scaffold are applied. At first, the
dry weight of the scaffold is measured as W0, and then the scaffold
is soaked in the double distilled water overnight. After that, the
scaffold is taken out from the water, and the moisture on its surface is
wiped off. Them the weight is measured as W1. Then, the water
absorption is

( W 1 - W 0 ) W 0 × 100 % . ##EQU00002##

[0034]In the other hand, the measurement of porosity is based on
Archimedes' Principle and use the pycnometer. At first, the pycnometer is
made full of water, and then the weight of above pycnometer is measured
as W1. In the other hand, the weight of the scaffold is measured as
WS. Subsequently, the scaffold is put into the pycnometer, and the
air is made out of the scaffold. After the pycnometer is refilled with
water, the weight is measured as W2. After that, the scaffold that
is full of water is taken out of the pycnometer and the weight of the
pycnometer at that time is measured as W3. Then, the porosity
(ε) is

[0035]The experimental result shows that the water absorption and the
porosity of the scaffold both increase as the poly(γ-glutamic
acid)-g-chondroitin sulfate copolymer content increases. This approves
that adding poly(γ-glutamic acid)-g-chondroitin sulfate copolymer
improves the water absorption and increase the porosity of the scaffold.

Cytotoxicity Test

[0036]In this embodiment of the present invention, the cytotoxicity test
is performed on the scaffold made of the porous composite biomaterials.
First, the scaffold is soaked in 70% alcohol. Then the scaffold is put on
the sterile bench and irradiated with ultraviolet ray for 22 hours.
Subsequently, the scaffold is washed and soaked with phosphate buffer
solution for three times under the radiation of ultraviolet ray and put
on the ninety-six wells culture plate. After that, the 3T3 fibroblasts
are inoculated onto the scaffold inside the ninety-six wells culture
plate with the inoculum density of 2×105 cells/scaffold and
standed 3 hours for the attachment of the cells onto the scaffold. Then
the scaffold is moved to a twelve wells culture plate, and adequate
amount of cell culture brothes are added into the plate. Subsequently,
the plate is put into the incubator for cell culture in 37° C.,
and the cell culture brothes are changed every two days. The cell culture
brothes are removed at the initial day and the every second day. Then the
scaffold is washed with phosphate buffer solution, and 40 μl/well
dimethyl sulfoxide is added into it. When dissolving completely, 200
μl of the solution is taken into the ninety six wells culture plate,
and its absorbance at the wavelength of 570 nm is recorded. The result is
shown as FIG. 9 which verifies the scaffold made of the porous composite
biomaterial of the present invention is non-cytotoxic.

Analysis of Glycosaminoglycan (GAG) and Collagen

[0037]In this embodiment of the present invention, the absorbance
properties of the complex for forming by pigments and glycosaminoglycan
(GAG) is utilized to make quantitative analysis, wherein the
glycosaminoglycan is a saccharide binding around the collagen. At first,
the chondrocytes are decomposed in the scaffold by papain, and then they
are dyed with 1,9-dimethylmethylene. The spectrophotometer is used then
to determine the contents which are usually the indicator of the quality
of the artificial cartilage. The applied method comprising hydrolyzing
the collagen with the existence of strong acid in high temperature;
releasing the hydroxyproline; dyeing with pigments; radiate with 550 nm
ray; recording the absorbance and ploting the standard curve; and
calculating the concentration of glycosaminoglycan and collagen. The
result is shown as Table 3.

[0038]The copolymer shown in Table 3 means the poly(γ-glutamic
acid)-g-chondroitin sulfate copolymer of the present invention. The
experimental result indicates that the glycosaminoglycan and the collagen
in the scaffold increase as time goes by. Then, comparing with the
samples of PCL, R10P90, and R30P70, it may be concluded that the more the
poly(γ-glutamic acid)-g-chondroitin sulfate copolymer contained in
the samples, the more the GAG and collagen secreted by cells. And this
will make the cells more active outside the human bodies.

[0039]In conclusion, the scaffold, made of the porous composite
biomaterials comprising poly(γ-glutamic acid)-g-chondroitin sulfate
copolymer and poly(ε-caprolactone), owns greater hydrophilicity,
cell adsorption ability, and degradability than the scaffold made of only
poly(ε-caprolactone). And further, the cell culture of
chondrocytes on the porous composite biomaterial also performs better
than only poly(ε-caprolactone), and filling the requirements more
for producing artificial cartilages in the field of biomaterial.

[0040]Embodiments of the invention are illustrated by way of example, and
not by way of limitation. The scope of the present invention should be
determined by the claims below. One skilled in the art may make further
modifications and adaptations without departing from the basic scope of
the present invention. Thus the modifications and adaptations are
belonging to the spirit of the teaching here and should be included among
the following claims.